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Coupled-column HPLC analysis of free urinary catecholamines using restricted access affinity precolumn and micro-particulate nonporous silica analytical column

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Summary

A coupled-column LC-method for fast, direct and routine quantitation of free parent catecholamines norepinephrine, epinephrine and dopamine in human urine has been developed. Direct injection of untreated samples followed by system-integrated, sample cleanup was achieved using a restricted-access affinity precolumn packed with a boronic acid-modified, copolymer support. Analytes were selectively extracted from the urine matrix at an alkaline pH by covalent affinity bonding. Desorption and transfer of the catecholamines to a nonporous, microparticulate-silica analytical column was by changing the pH of the mobile phase to an acidic value.

Separation was by ion-pair RP-HPLC under aqueous conditions without addition of organic modifier. Analytes were detected by their natural fluorescence. Limits of quantitation were 5.57, 1.75 and 36.81 pmol for norepinephrine, epinephrine and dopamine, respectively. Urine levels could be quantified with a precision of about 2%. Mean recoveries of norepinephrine, epinephrine and dopamine were 98.18, 102.0 and 101.12%. Using a nonporous packing in the analytical column, analytical times and solvent consumption were reduced considerably compared to conventional porous silica columns.

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Rudolphi, A., Boos, K.-. & Seidel, D. Coupled-column HPLC analysis of free urinary catecholamines using restricted access affinity precolumn and micro-particulate nonporous silica analytical column. Chromatographia 41, 645–650 (1995). https://doi.org/10.1007/BF02267798

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Key Words

  • Coupled-column liquid chromatography
  • Affinity chromatography
  • Restricted access material
  • Nonporous microparticulate silica
  • Catecholamines